Aglycosylated Fc-containing polypeptides with cysteine substitutions

ABSTRACT

Provided herein are IL-2 muteins and IL-2 mutein Fc-fusion molecules that preferentially expand and activate T regulatory cells and are amenable to large scale production. Also provided herein are variant human IgG1 Fc molecules lacking or with highly reduced effector function and high stability despite lacking glycosylation at N297. Also, provided herein are linker peptides that are glycosylated when expressed in mammalian cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/784,669, filed Mar. 14, 2013. The above-identified application isincorporated herein by reference.

REFERENCE TO THE SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format via EFS-Web. The Sequence Listing is provided as atext file entitled A-1892-US-NP_ST25.txt, created Mar. 3, 2014, which is40,960 bytes in size. The information in the electronic format of theSequence Listing is incorporated herein by reference in its entirety.

BACKGROUND

IL-2 binds three transmembrane receptor subunits: IL-2Rβ and IL-2Rγwhich together activate intracellular signaling events upon IL-2binding, and CD25 (IL-2Rα) which serves to stabilize the interactionbetween IL-2 and IL-2Rβγ. The signals delivered by IL-2Rβγ include thoseof the PI3-kinase, Ras-MAP-kinase, and STAT5 pathways.

T cells require expression of CD25 to respond to the low concentrationsof IL-2 that typically exist in tissues. T cells that express CD25include both FOXP3+ regulatory T cells (Treg cells), which are essentialfor suppressing autoimmune inflammation, and FOXP3− T cells that havebeen activated to express CD25. FOXP3−CD25+ T effector cells (Teff) maybe either CD4+ or CD8+ cells, both of which may contribute toinflammation, autoimmunity, organ graft rejection, or graft-versus-hostdisease. IL-2-stimulated STAT5 signaling is crucial for normal T-regcell growth and survival and for high FOXP3 expression.

In co-owned WO 2010/085495, we describe the use of IL-2 muteins topreferentially expand or stimulate Treg cells. When administered to asubject, the effect on Treg cells is useful for treating inflammatoryand autoimmune diseases. Although the IL-2 muteins described therein areuseful for expanding Treg over Teff cells in vivo, it was desirable tocreate IL-2 muteins that had optimal attributes for a human therapeutic.

SUMMARY

Described herein are IL-2 muteins that are amenable to high-yieldmanufacturability and have optimized pharmacological activity. In theeffort to produce an exemplary IL-2 mutein-based human therapeutic, anumber of unexpected and unpredictable observations occurred. The IL-2muteins described herein are the result of that effort.

The IL-2 muteins described herein have a minimal numbers of alterationsto IL-2, thereby decreasing the likelihood of creating an immuneresponse against the IL-2 mutein and/or endogenous IL-2, yet maintainTreg preferential expansion and activation. Moreover, in certainembodiments, the IL-2 mutein is fused to a molecule, e.g. an antibodyFc, that increases the serum half-life when administered to a subject.IL-2 muteins have a short serum half-life (3 to 5 hrs for sub-cutaneousinjection). Exemplary IL-2 mutein Fc fusions described herein have ahalf-life in humans of at least 1 day, at least 3 days, at least 5 days,at least 10 days, at least 15 days, at least 20 days, or at least 25days. This effect on the pharmacokinetics of the IL-2 muteins allows fordecreased or less frequent dosing of the IL-2 mutein therapeutic.

Moreover, when creating a pharmaceutical large molecule, considerationmust be made for the ability to produce the large molecule in largequantities, while minimizing aggregation and maximizing the stability ofthe molecule. The IL-2 mutein Fc-fusion molecules demonstrate suchattributes.

Additionally, in certain embodiments, the IL-2 mutein Fc-fusion proteincontains an IgG1 Fc region. When it is desirable to abolish the effectorfunctions of IgG1 (e.g., ADCC activity), it was found that mutation ofthe asparagine at position 297 to glycine (N297G; EU numbering scheme)provided greatly improved purification efficiency and biophysicalproperties over other mutations that lead to an aglycosylation IgG1 Fc.In preferred embodiments, cysteines are engineered into the Fc to allowdisulfide bonds, which increased stability of the aglycosylatedFc-containing molecule. The usefulness of the aglycosylated Fc goesbeyond the IL-2 mutein Fc-fusion context. Thus, provided herein areFc-containing molecules, Fc-fusions and antibodies, comprising a N297Gsubstitution and optionally substitution of one or more additionalresidues to cysteine.

Another aspect of the invention includes glycosylated peptide linkers.Preferred linker peptides that are amenable to N-glycosylation includeGGNGT (SEQ ID NO:6) or YGNGT (SEQ ID NO:7).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 In a short term stimulation assay, homodimerization by fusion tothe C-terminus of IgG-Fc does not alter the activity of IL-2 muteinswith reduced potency and with high affinity for CD25.

FIG. 2A and FIG. 2B IL-2 muteins with the indicated mutations and fusedto the C-terminus of one side of an Fc-heterodimer were tested for theirability to stimulate STAT5 phosphorylation in T cells. These muteinsalso contained three mutations conferring high affinity for CD25 (V69A,N71R, Q74P). Their activity was compared to three forms of IL-2 withoutFc fusion (open symbols): WT IL-2, HaWT (high affinity for CD25) (N29S,Y31H, K35R, T37A, K48E, V69A, N71R, Q74P), and HaD (high affinity forCD25 and reduced signaling activity) (N29S, Y31H, K35R, T37A, K48E,V69A, N71R, Q74P, N88D). Phospho-STAT5 responses are shown for gatedFOXP3+ CD4+ and FOXP3−CD4+ T cells.

FIG. 3 Proliferation of T cell subsets in response to titrations of IL-2muteins fused to Fc-heterodimer. Activity of fusion proteins wascompared to three forms of IL-2 without Fc fusion (open symbols): WTIL-2, HaWT (high affinity for CD25) (N29S, Y31H, K35R, T37A, K48E, V69A,N71R, Q74P), and HaD (high affinity for CD25 and reduced signalingactivity) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D)

FIG. 4 Proliferation of NK cells in response to titrations of IL-2muteins fused to Fc-heterodimer. Activity of fusion proteins wascompared to three forms of IL-2 without Fc fusion (open symbols): WTIL-2, HaWT (high affinity for CD25) (N29S, Y31H, K35R, T37A, K48E, V69A,N71R, Q74P), and HaD (high affinity for CD25 and reduced signalingactivity) (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D)

FIG. 5 Proliferation of T cell subsets in response to titrations of IL-2muteins fused to Fc-homodimer N297G. Activity of Fc.muteins was comparedto WT IL-2 (open circles) and Fc.WT (closed circles). Mutations thatconfer high affinity for CD25 (HaMut1) were V69A and 074P.

FIG. 6 Proliferation of NK cells in response to titrations of IL-2muteins fused to Fc-homodimer N297G. Activity of Fc.muteins was comparedto WT IL-2 (open circles) and Fc.WT (closed circles).

FIG. 7A and FIG. 7B Fc.IL-2 muteins without mutations that confer highaffinity for CD25 promote Treg expansion and FOXP3 upregulation inhumanized mice.

FIG. 8 Low weekly doses (0.5 μg per animal) of Fc.IL-2 muteins promoteTreg expansion and FOXP3 upregulation in humanized mice, with betteractivity observed for Fc.V91K relative to Fc.N88D and Fc.WT.

FIG. 9 Fc.V91K and Fc.N88D persist on the surface of activated T cellsthrough association with CD25.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All references cited within the body of this specification are expresslyincorporated by reference in their entirety.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, tissue culture and transformation, protein purification, etc.Enzymatic reactions and purification techniques may be performedaccording to the manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The following proceduresand techniques may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thespecification. See, e.g., Sambrook et al., 2001, Molecular Cloning: ALaboratory Manuel, 3^(rd) ed., Cold Spring Harbor Laboratory Press, coldSpring Harbor, N.Y., which is incorporated herein by reference for anypurpose. Unless specific definitions are provided, the nomenclature usedin connection with, and the laboratory procedures and techniques of,analytic chemistry, organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques may be used for chemical synthesis, chemicalanalyses, pharmaceutical preparation, formulation, and delivery andtreatment of patients.

IL-2

The IL-2 muteins described herein are variants of wild-type human IL-2.As used herein, “wild-type human IL-2,” “wild-type IL-2,” or “WT IL-2”shall mean the polypeptide having the following amino acid sequence:

(SEQ ID NO: 2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFXQSIISTLT Wherein X is C, S, V, or A.

Variants may contain one or more substitutions, deletions, or insertionswithin the wild-type IL-2 amino acid sequence. Residues are designatedherein by the one letter amino acid code followed by the IL-2 amino acidposition, e.g., K35 is the lysine residue at position 35 of SEQ ID NO:2. Substitutions are designated herein by the one letter amino acid codefollowed by the IL-2 amino acid position followed by the substitutingone letter amino acid code., e.g., K35A is a substitution of the lysineresidue at position 35 of SEQ ID NO:2 with an alanine residue.

IL-2 Muteins

Provided herein are human IL-2 muteins that preferentially stimulate Tregulatory (Treg) cells. As used herein “preferentially stimulates Tregulatory cells” means the mutein promotes the proliferation, survival,activation and/or function of CD3+FoxP3+ T cells over CD3+FoxP3− Tcells. Methods of measuring the ability to preferentially stimulateTregs can be measured by flow cytometry of peripheral blood leukocytes,in which there is an observed increase in the percentage of FOXP3+CD4+ Tcells among total CD4+ T cells, an increase in percentage of FOXP3+CD8+T cells among total CD8+ T cells, an increase in percentage of FOXP3+ Tcells relative to NK cells, and/or a greater increase in the expressionlevel of CD25 on the surface of FOXP3+ T cells relative to the increaseof CD25 expression on other T cells. Preferential growth of Treg cellscan also be detected as increased representation of demethylated FOXP3promoter DNA (i.e. the Treg-specific demethylated region, or TSDR)relative to demethylated CD3 genes in DNA extracted from whole blood, asdetected by sequencing of polymerase chain reaction (PCR) products frombisulfite-treated genomic DNA (J. Sehouli, et al. 2011. Epigenetics 6:2,236-246).

IL-2 muteins that preferentially stimulate Treg cells increase the ratioof CD3+ FoxP3+ T cells over CD3+FoxP3− T cells in a subject or aperipheral blood sample at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 100%, atleast 150%, at least 200%, at least 300%, at least 400%, at least 500%,at least 600%, at least 700%, at least 800%, at least 900%, or at least1000%.

Preferred IL-2 muteins include, but are not limited to, IL-2 muteinscomprising V91K or N88D substitution in the amino acid sequence setforth in SEQ ID NO:2. An exemplary IL-2 mutein is set forth in SEQ IDNO:1. Particularly preferred is the amino acid sequence set forth in SEQID NO:1 comprising a C125A substitution. Although it may be advantageousto reduce the number of further mutations to the wild-type IL-2sequence, the invention includes IL-2 muteins having truncations oradditional insertions, deletions, or substitutions in addition to theV91K or N88D substitution, provided that said muteins maintain theactivity of preferentially simulating Tregs. Thus, embodiments includeIL-2 muteins that preferentially stimulate Treg cells and comprise anamino acid sequence having a V91K or N88D that is at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical to the aminoacid sequence set forth in SEQ ID NO:2. In particularly preferredembodiments, such IL-2 muteins comprises an amino acid sequence that isat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the amino acid sequence set forth in SEQ ID NO:2.

For amino acid sequences, sequence identity and/or similarity isdetermined by using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman, 1988, Proc. Nat.Acad. Sci. U.S.A. 85:2444, computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al., 1984,Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, 1987, J. Mol.Evol. 35:351-360; the method is similar to that described by Higgins andSharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al.,1997, Nucleic Acids Res. 25:3389-3402; and Karin et al., 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLASTprogram is the WU-BLAST-2 program which was obtained from Altschul etal., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2parameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to about 22 bits.

While the site or region for introducing an amino acid sequencevariation may be predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed IL-2 mutein screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants may be done using assays describedherein, for example.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about one (1) to about twenty (20)amino acid residues, although considerably larger insertions may betolerated. Deletions range from about one (1) to about twenty (20) aminoacid residues, although in some cases deletions may be much larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative or variant. Generally these changesare done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of the antigenbinding protein. However, larger changes may be tolerated in certaincircumstances. Conservative substitutions are generally made inaccordance with the following chart depicted as TABLE 1.

TABLE 1 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro His Asn, Gln IleLeu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr,Trp Ser Thr Thr Ser Trp Tyr, Phe Tyr Trp, Phe Val Ile, LeuSubstantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTABLE 1. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g., seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the IL-2 mutein as needed. Alternatively, the variantmay be designed such that the biological activity of the IL-2 mutein isaltered. For example, glycosylation sites may be altered or removed asdiscussed herein.

IL-2 Muteins Having Extended Serum Half-life

Because the IL-2 muteins provided herein preferentially expand Tregsover, for example Teff or NK cells, it is expected that the safetyprofile when administered to a patient will differ from that ofwild-type IL-2 or PROLEUKIN. Side-effects associated with wild-type IL-2or PROLEUKIN include flu-like symptoms, chills/rigor, arthralgia, fever,rash, pruritus, injection site reactions, hypotension, diarrhea, nausea,anxiety, confusion, and depression. The IL-2 muteins provided herein maybe altered to include or fused to molecules that extend the serumhalf-life of the mutein without increasing the risk that such half-lifeextension would increase the likelihood or the intensity of aside-effect or adverse event in a patient. Subcutaneous dosing of suchan extended serum half-life mutein may allow for prolonged targetcoverage with lower systemic maximal exposure (C_(max)). Extended serumhalf-life may allow a lower or less frequent dosing regimen of themutein.

The serum half-life of the IL-2 muteins provided herein may be extendedby essentially any method known in the art. Such methods includealtering the sequence of the IL-2 mutein to include a peptide that bindsto the neonatal Fcγ receptor or bind to a protein having extended serumhalf-life, e.g., IgG or human serum albumin. In other embodiments, theIL-2 mutein is fused to a polypeptide that confers extended half-life onthe fusion molecule. Such polypeptides include an IgG Fc or otherpolypeptides that bind to the neonatal Fcγ receptor, human serumalbumin, or polypeptides that bind to a protein having extended serumhalf-life. In preferred embodiments, the IL-2 mutein is fused to an IgGFc molecule.

The IL-2 mutein may be fused to the N-terminus or the C-terminus of theIgG Fc region. As shown in the Examples, fusion to the C-terminus of theIgG Fc region maintains the IL-2 mutein activity to a greater extentthan when fused to the N-terminus of the IgG Fc.

One embodiment of the present invention is directed to a dimercomprising two Fc-fusion polypeptides created by fusing an IL-2 muteinto the Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain bonds form between the Fc moieties to yield the dimer.

The term “Fc polypeptide” or “Fc region” as used herein includes nativeand mutein forms of polypeptides derived from the Fc region of anantibody. Truncated forms of such polypeptides containing the hingeregion that promotes dimerization also are included. In certainembodiments, the Fc region comprises an antibody CH2 and CH3 domain.Along with extended serum half-life, fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns. Preferred Fc regions are derived from human IgG, which includesIgG1, IgG2, IgG3, and IgG4. Herein, specific residues within the Fc areidentified by position. All Fc positions are based on the EU numberingscheme.

One of the functions of the Fc portion of an antibody is to communicateto the immune system when the antibody binds its target. This isconsidered “effector function.” Communication leads toantibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP), and/or complement dependent cytotoxicity(CDC). ADCC and ADCP are mediated through the binding of the Fc to Fcreceptors on the surface of cells of the immune system. CDC is mediatedthrough the binding of the Fc with proteins of the complement system,e.g., C1q.

The IgG subclasses vary in their ability to mediate effector functions.For example, IgG1 is much superior to IgG2 and IgG4 at mediating ADCCand CDC. Thus, in embodiments wherein effector function is undesirable,an IgG2 Fc would be preferred. IgG2 Fc-containing molecules, however,are known to be more difficult to manufacture and have less attractivebiophysical properties, such as a shorter half-life, as compared to IgG1Fc-containing molecules.

The effector function of an antibody can be increased, or decreased, byintroducing one or more mutations into the Fc. Embodiments of theinvention include IL-2 mutein Fc fusion proteins having an Fc engineeredto increase effector function (U.S. Pat. No. 7,317,091 and Strohl, Curr.Opin. Biotech., 20:685-691, 2009; both incorporated herein by referencein its entirety). Exemplary IgG1 Fc molecules having increased effectorfunction include those having the following substitutions:

S239D/I332E S239D/A330S/I332E S239D/A330L/I332E S298A/D333A/K334AP247I/A339D P247I/A339Q D280H/K290S D280H/K290S/S298D D280H/K290S/S298VF243L/R292P/Y300L F243L/R292P/Y300L/P396L F243L/R292P/Y300L/V305I/P396LG236A/S239D/I332E K326A/E333A K326W/E333S K290E/S298G/T299AK290N/S298G/T299A K290E/S298G/T299A/K326E K290N/S298G/T299A/K326E

Another method of increasing effector function of IgG Fc-containingproteins is by reducing the fucosylation of the Fc. Removal of the corefucose from the biantennary complex-type oligosachharides attached tothe Fc greatly increased ADCC effector function without altering antigenbinding or CDC effector function. Several ways are known for reducing orabolishing fucosylation of Fc-containing molecules, e.g., antibodies.These include recombinant expression in certain mammalian cell linesincluding a FUT8 knockout cell line, variant CHO line Lec13, rathybridoma cell line YB2/0, a cell line comprising a small interferingRNA specifically against the FUT8 gene, and a cell line coexpressingβ-1,4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II.Alternatively, the Fc-containing molecule may be expressed in anon-mammalian cell such as a plant cell, yeast, or prokaryotic cell,e.g., E. coli.

In preferred embodiments of the invention, IL-2 mutein Fc-fusionproteins comprise an Fc engineered to decrease effector function.Exemplary Fc molecules having decreased effector function include thosehaving the following substitutions:

N297A or N297Q (IgG1) L234A/L235A (IgG1) V234A/G237A (IgG2)L235A/G237A/E318A (IgG4) H268Q/V309L/A330S/A331S (IgG2)C220S/C226S/C229S/P238S (IgG1) C226S/C229S/E233P/L234V/L235A (IgG1)L234F/L235E/P331S (IgG1) S267E/L328F (IgG1)

It is known that human IgG1 has a glycosylation site at N297 (EUnumbering system) and glycosylation contributes to the effector functionof IgG1 antibodies. An exemplary IgG1 sequence is provided in SEQ IDNO:3. Groups have mutated N297 in an effort to make aglycosylatedantibodies. The mutations have focuses on substituting N297 with aminoacids that resemble asparagine in physiochemical nature such asglutamine (N297Q) or with alanine (N297A) which mimics asparagineswithout polar groups.

As used herein, “aglycosylated antibody” or “aglycosylated fc” refers tothe glycosylation status of the residue at position 297 of the Fc. Anantibody or other molecule may contain glycosylation at one or moreother locations but may still be considered an aglycosylated antibody oraglcosylated Fc-fusion protein.

In our effort to make an effector functionless IgG1 Fc, it wasdiscovered that mutation of amino acid N297 of human IgG1 to glycine,i.e., N297G, provides far superior purification efficiency andbiophysical properties over other amino acid substitutions at thatresidue. See Example 8. Thus, in preferred embodiments, the IL-2 muteinFc-fusion protein comprises a human IgG1 xFc having a N297Gsubstitution. The Fc comprising the N297G substitution is useful in anycontext wherein a molecule comprises a human IgG1 Fc, and is not limitedto use in the context of an IL-2 mutein Fc-fusion. In certainembodiments, an antibody comprises the Fc having a N297G substitution.

An Fc comprising a human IgG1 Fc having the N297G mutation may alsocomprise further insertions, deletions, and substitutions. In certainembodiments the human IgG1 Fc comprises the N297G substitution and is atleast 90% identical, at least 91% identical, at least 92% identical, atleast 93% identical, at least 94% identical, at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the amino acid sequence set forth in SEQ IDNO:3. In a particularly preferred embodiment, the C-terminal lysineresidue is substituted or deleted. The amino acid sequence of human IgG1comprising the N297G substitution and deletion of the C-terminal lysineis set forth in SEQ ID NO:4.

A glycosylated IgG1 Fc-containing molecules were shown to be less stablethan glycosylated IgG1 Fc-containing molecules. The Fc region may befurther engineered to increase the stability of the aglycosylatedmolecule. In some embodiments, one or more amino acids are substitutedto cysteine so to form di-sulfide bonds in the dimeric state. ResiduesV259, A287, R292, V302, L306, V323, or 1332 of the amino acid sequenceset forth in SEQ ID NO:3 may be substituted with cysteine. In preferredembodiments, specific pairs of residues are substitution such that theypreferentially form a di-sulfide bond with each other, thus limiting orpreventing di-sulfide bond scrambling. Preferred pairs include, but arenot limited to, A287C and L306C, V259C and L306C, R292c and V302C, andV323C and 1332C.

Provided herein are Fc-containing molecules wherein one or more ofresidues V259, A287, R292, V302, L306, V323, or 1332 are substitutedwith cysteine. Preferred Fc-containing molecules include thosecomprising A287C and L306C, V259C and L306C, R292c and V302C, or V323Cand 1332C substitutions.

Additional mutations that may be made to the IgG1 Fc include thosefacilitate heterodimer formation amongst Fc-containing polypeptides. Insome embodiments, Fc region is engineering to create “knobs” and “holes”which facilitate heterodimer formation of two different Fc-containingpolypeptide chains when co-expressed in a cell. U.S. Pat. No. 7,695,963.In other embodiments, the Fc region is altered to use electrostaticsteering to encourage heterodimer formation while discouraging homodimerformation of two different Fc-containing polypeptide when co-expressedin a cell. WO 09/089,004, which is incorporated herein by reference inits entirety. Preferred heterodimeric Fc include those wherein one chainof the Fc comprises D399K and E356K substitutions and the other chain ofthe Fc comprises K409D and K392D substitutions. In other embodiments,one chain of the Fc comprises D399K, E356K, and E357K substitutions andthe other chain of the Fc comprises K409D, K392D, and K370Dsubstitutions.

In certain embodiments, it may be advantageous for the IL-2 muteinFc-fusion protein to be monomeric, i.e., contain only a single IL-2mutein molecule. In such embodiments, the Fc-region of the fusionprotein may contain one or more mutations that facilitate heterodimerformation. The fusion protein is co-expressed with an Fc-region havingreciprocal mutations to those in the IL-2 mutein Fc-fusion polypeptidebut lacking an IL-2 mutein. When the heterodimer of the twoFc-containing polypeptides forms, the resulting protein comprises only asingle IL-2 mutein.

Another method of creating a monomeric IL-2 mutein Fc-fusion protein isfusing the IL-2 mutein to a monomeric Fc, i.e., an Fc region that doesnot dimerize. Stable monomeric Fcs comprise mutations that discouragedimerization and that stabilize the molecule in the monomeric form.Preferred monomeric Fcs are disclosed in WO 2011/063348, which isincorporated herein by reference in its entirety. In certainembodiments, IL-2 mutein Fc fusion proteins comprise an Fc comprisingnegatively charged amino acids at positions 392 and 409 along with athreonine substitution at Y349, L351, L368, V397, L398, F405, or Y407.

In certain embodiments, the IL-2 mutein Fc-fusion protein comprises alinker between the Fc and the IL-2 mutein. Many different linkerpolypeptides are known in the art and may be used in the context of anIL-2 mutein Fc-fusion protein. In preferred embodiments, the IL-2 muteinFc-fusion protein comprises one or more copies of a peptide consistingof GGGGS (SEQ ID NO:5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7)between the Fc and the IL-2 mutein. In some embodiments, the polypeptideregion between the Fc region and the IL-2 mutein region comprises asingle copy of GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQID NO: 7). As shown herein, the linkers GGNGT (SEQ ID NO: 6) or YGNGT(SEQ ID NO: 7) are glycosylated when expressed in the appropriate cellsand such glycosylation may help stabilize the protein in solution and/orwhen administered in vivo. Thus, in certain embodiments, an IL-2 muteinfusion protein comprises a glycosylated linker between the Fc region andthe IL-2 mutein region.

It is contemplated that the glycosylated linker may be useful whenplaced in the context of a polypeptide. Provided herein are polypeptidescomprising GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7) inserted intothe amino acid sequence of the polypeptide or replacing one or moreamino acids within the amino acid sequence of the polypeptide. Inpreferred embodiments, GGNGT (SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7) isinserted into a loop of the polypeptides tertiary structure. In otherembodiments, one or more amino acids of a loop are replaced with GGNGT(SEQ ID NO: 6) or YGNGT (SEQ ID NO: 7).

The C-terminal portion of the Fc and/or the amino terminal portion ofthe IL-2 mutein may contain one or more mutations that alter theglycosylation profile f the IL-2 mutein Fc-fusion protein when expressedin mammalian cells. In certain embodiments, the IL-2 mutein furthercomprises a T3 substitution, e.g., T3N or T3A. The IL-2 mutein mayfurther comprise an S5 substitution, such as S5T

Covalent modifications of IL-2 mutein and IL-2 mutein Fc-fusion proteinsare included within the scope of this invention, and are generally, butnot always, done post-translationally. For example, several types ofcovalent modifications of the IL-2 mutein or IL-2 mutein Fc-fusionprotein are introduced into the molecule by reacting specific amino acidresidues of the IL-2 mutein or IL-2 mutein Fc-fusion protein with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantigen binding proteins to a water-insoluble support matrix or surfacefor use in a variety of methods. Commonly used crosslinking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, 1983, pp. 79-86),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification of the IL-2 mutein or IL-2 muteinFc-fusion protein included within the scope of this invention comprisesaltering the glycosylation pattern of the protein. As is known in theart, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the IL-2 mutein or IL-2 muteinFc-fusion protein may be conveniently accomplished by altering the aminoacid sequence such that it contains one or more of the above-describedtri-peptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues to the starting sequence (for O-linkedglycosylation sites). For ease, the IL-2 mutein or IL-2 mutein Fc-fusionprotein amino acid sequence is preferably altered through changes at theDNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theIL-2 mutein or IL-2 mutein Fc-fusion protein is by chemical or enzymaticcoupling of glycosides to the protein. These procedures are advantageousin that they do not require production of the protein in a host cellthat has glycosylation capabilities for N- and O-linked glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine, (b) free carboxyl groups, (c) free sulfhydrylgroups such as those of cysteine, (d) free hydroxyl groups such as thoseof serine, threonine, or hydroxyproline, (e) aromatic residues such asthose of phenylalanine, tyrosine, or tryptophan, or (f) the amide groupof glutamine. These methods are described in WO 87/05330 published Sep.11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp.259-306.

Removal of carbohydrate moieties present on the starting IL-2 mutein orIL-2 mutein Fc-fusion protein may be accomplished chemically orenzymatically. Chemical deglycosylation requires exposure of the proteinto the compound trifluoromethanesulfonic acid, or an equivalentcompound. This treatment results in the cleavage of most or all sugarsexcept the linking sugar (N-acetylglucosamine or N-acetylgalactosamine),while leaving the polypeptide intact. Chemical deglycosylation isdescribed by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 andby Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage ofcarbohydrate moieties on polypeptides can be achieved by the use of avariety of endo- and exo-glycosidases as described by Thotakura et al.,1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycinblocks the formation of protein-N-glycoside linkages.

Another type of covalent modification of the IL-2 mutein or IL-2 muteinFc-fusion protein comprises linking the IL-2 mutein or IL-2 muteinFc-fusion protein to various nonproteinaceous polymers, including, butnot limited to, various polyols such as polyethylene glycol,polypropylene glycol or polyoxyalkylenes, in the manner set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337. In addition, amino acid substitutions may be made in variouspositions within the IL-2 mutein or IL-2 mutein Fc-fusion protein tofacilitate the addition of polymers such as PEG. Thus, embodiments ofthe invention include PEGylated IL-2 muteins and IL-2 mutein Fc-fusionproteins. Such PEGylated proteins may have increase increased half-lifeand/or reduced immunogenicity over the non-PEGylated proteins.

Polynucleotides Encoding IL-2 Muteins and IL-2 Mutein Fc-Fusion Proteins

Encompassed within the invention are nucleic acids encoding IL-2 muteinsand IL-2 mutein Fc-fusion proteins. Aspects of the invention includepolynucleotide variants (e.g., due to degeneracy) that encode the aminoacid sequences described herein. In preferred embodiments, thepolypeptide encoded by the isolated nucleic acid is a component of anIL-2 mutein Fc-fusion protein.

Nucleotide sequences corresponding to the amino acid sequences describedherein, to be used as probes or primers for the isolation of nucleicacids or as query sequences for database searches, can be obtained by“back-translation” from the amino acid sequences. The well-knownpolymerase chain reaction (PCR) procedure can be employed to isolate andamplify a DNA sequence encoding IL-2 muteins and IL-2 mutein Fc-fusionprotein. Oligonucleotides that define the desired termini of thecombination of DNA fragments are employed as 5′ and 3′ primers. Theoligonucleotides can additionally contain recognition sites forrestriction endonucleases, to facilitate insertion of the amplifiedcombination of DNA fragments into an expression vector. PCR techniquesare described in Saiki et al., Science 239:487 (1988); Recombinant DNAMethodology, Wu et al., eds., Academic Press, Inc., San Diego (1989),pp. 189-196; and PCR Protocols: A Guide to Methods and Applications,Innis et. al., eds., Academic Press, Inc. (1990).

Nucleic acid molecules of the invention include DNA and RNA in bothsingle-stranded and double-stranded form, as well as the correspondingcomplementary sequences. An “isolated nucleic acid” is a nucleic acidthat has been separated from adjacent genetic sequences present in thegenome of the organism from which the nucleic acid was isolated, in thecase of nucleic acids isolated from naturally-occurring sources. In thecase of nucleic acids synthesized enzymatically from a template orchemically, such as PCR products, cDNA molecules, or oligonucleotidesfor example, it is understood that the nucleic acids resulting from suchprocesses are isolated nucleic acids. An isolated nucleic acid moleculerefers to a nucleic acid molecule in the form of a separate fragment oras a component of a larger nucleic acid construct. In one preferredembodiment, the nucleic acids are substantially free from contaminatingendogenous material. The nucleic acid molecule has preferably beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

The variants according to the invention are ordinarily prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the IL-2 muteinor IL-2 mutein Fc-fusion protein, using cassette or PCR mutagenesis orother techniques well known in the art, to produce DNA encoding thevariant, and thereafter expressing the recombinant DNA in cell cultureas outlined herein. However, IL-2 muteins and IL-2 mutein Fc-fusion maybe prepared by in vitro synthesis using established techniques. Thevariants typically exhibit the same qualitative biological activity asthe naturally occurring analogue, e.g., Treg expansion, althoughvariants can also be selected which have modified characteristics aswill be more fully outlined below.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode IL-2 muteins and IL-2 mutein Fc-fusion proteins ofthe present invention. Thus, having identified a particular amino acidsequence, those skilled in the art could make any number of differentnucleic acids, by simply modifying the sequence of one or more codons ina way which does not change the amino acid sequence of the encodedprotein.

The present invention also provides expression systems and constructs inthe form of plasmids, expression vectors, transcription or expressioncassettes which comprise at least one polynucleotide as above. Inaddition, the invention provides host cells comprising such expressionsystems or constructs.

Typically, expression vectors used in any of the host cells will containsequences for plasmid maintenance and for cloning and expression ofexogenous nucleotide sequences. Such sequences, collectively referred toas “flanking sequences” in certain embodiments will typically includeone or more of the following nucleotide sequences: a promoter, one ormore enhancer sequences, an origin of replication, a transcriptionaltermination sequence, a complete intron sequence containing a donor andacceptor splice site, a sequence encoding a leader sequence forpolypeptide secretion, a ribosome binding site, a polyadenylationsequence, a polylinker region for inserting the nucleic acid encodingthe polypeptide to be expressed, and a selectable marker element. Eachof these sequences is discussed below.

Optionally, the vector may contain a “tag”-encoding sequence, i.e., anoligonucleotide molecule located at the 5′ or 3′ end of the IL-2 muteinsor IL-2 mutein Fc-fusion protein coding sequence; the oligonucleotidesequence encodes polyHis (such as hexaHis (SEQ ID NO: 21)), or another“tag” such as FLAG, HA (hemaglutinin influenza virus), or myc, for whichcommercially available antibodies exist. This tag is typically fused tothe polypeptide upon expression of the polypeptide, and can serve as ameans for affinity purification or detection of the IL-2 mutein from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified IL-2 muteins and IL-2 mutein Fc-fusion proteins by variousmeans such as using certain peptidases for cleavage.

Flanking sequences may be homologous (i.e., from the same species and/orstrain as the host cell), heterologous (i.e., from a species other thanthe host cell species or strain), hybrid (i.e., a combination offlanking sequences from more than one source), synthetic or native. Assuch, the source of a flanking sequence may be any prokaryotic oreukaryotic organism, any vertebrate or invertebrate organism, or anyplant, provided that the flanking sequence is functional in, and can beactivated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Whether all or only a portion of the flanking sequence is known, it maybe obtained using polymerase chain reaction (PCR) and/or by screening agenomic library with a suitable probe such as an oligonucleotide and/orflanking sequence fragment from the same or another species. Where theflanking sequence is not known, a fragment of DNA containing a flankingsequence may be isolated from a larger piece of DNA that may contain,for example, a coding sequence or even another gene or genes. Isolationmay be accomplished by restriction endonuclease digestion to produce theproper DNA fragment followed by isolation using agarose gelpurification, Qiagen® column chromatography (Chatsworth, Calif.), orother methods known to the skilled artisan. The selection of suitableenzymes to accomplish this purpose will be readily apparent to one ofordinary skill in the art.

An origin of replication is typically a part of those prokaryoticexpression vectors purchased commercially, and the origin aids in theamplification of the vector in a host cell. If the vector of choice doesnot contain an origin of replication site, one may be chemicallysynthesized based on a known sequence, and ligated into the vector. Forexample, the origin of replication from the plasmid pBR322 (New EnglandBiolabs, Beverly, Mass.) is suitable for most gram-negative bacteria,and various viral origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitus virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, the origin ofreplication component is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it alsocontains the virus early promoter).

A transcription termination sequence is typically located 3′ to the endof a polypeptide coding region and serves to terminate transcription.Usually, a transcription termination sequence in prokaryotic cells is aG-C rich fragment followed by a poly-T sequence. While the sequence iseasily cloned from a library or even purchased commercially as part of avector, it can also be readily synthesized using methods for nucleicacid synthesis such as those described herein.

A selectable marker gene encodes a protein necessary for the survivaland growth of a host cell grown in a selective culture medium. Typicalselection marker genes encode proteins that (a) confer resistance toantibiotics or other toxins, e.g., ampicillin, tetracycline, orkanamycin for prokaryotic host cells; (b) complement auxotrophicdeficiencies of the cell; or (c) supply critical nutrients not availablefrom complex or defined media. Specific selectable markers are thekanamycin resistance gene, the ampicillin resistance gene, and thetetracycline resistance gene. Advantageously, a neomycin resistance genemay also be used for selection in both prokaryotic and eukaryotic hostcells.

Other selectable genes may be used to amplify the gene that will beexpressed. Amplification is the process wherein genes that are requiredfor production of a protein critical for growth or cell survival arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells. Examples of suitable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thyrnidinekinase genes. Mammalian cell transformants are placed under selectionpressure wherein only the transformants are uniquely adapted to surviveby virtue of the selectable gene present in the vector. Selectionpressure is imposed by culturing the transformed cells under conditionsin which the concentration of selection agent in the medium issuccessively increased, thereby leading to the amplification of both theselectable gene and the DNA that encodes another gene, such as an IL-2mutein or IL-2 mutein Fc-fusion protein. As a result, increasedquantities of a polypeptide such as an IL-2 mutein or IL-2 muteinFc-fusion protein are synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed. In certain embodiments, one or more coding regions may beoperably linked to an internal ribosome binding site (IRES), allowingtranslation of two open reading frames from a single RNA transcript.

In some cases, such as where glycosylation is desired in a eukaryotichost cell expression system, one may manipulate the various pre- orprosequences to improve glycosylation or yield. For example, one mayalter the peptidase cleavage site of a particular signal peptide, or addprosequences, which also may affect glycosylation. The final proteinproduct may have, in the −1 position (relative to the first amino acidof the mature protein) one or more additional amino acids incident toexpression, which may not have been totally removed. For example, thefinal protein product may have one or two amino acid residues found inthe peptidase cleavage site, attached to the amino-terminus.Alternatively, use of some enzyme cleavage sites may result in aslightly truncated form of the desired polypeptide, if the enzyme cutsat such area within the mature polypeptide.

Expression and cloning vectors of the invention will typically contain apromoter that is recognized by the host organism and operably linked tothe molecule encoding the IL-2 mutein or IL-2 mutein Fc-fusion protein.Promoters are untranscribed sequences located upstream (i.e., 5′) to thestart codon of a structural gene (generally within about 100 to 1000 bp)that control transcription of the structural gene. Promoters areconventionally grouped into one of two classes: inducible promoters andconstitutive promoters. Inducible promoters initiate increased levels oftranscription from DNA under their control in response to some change inculture conditions, such as the presence or absence of a nutrient or achange in temperature. Constitutive promoters, on the other hand,uniformly transcribe gene to which they are operably linked, that is,with little or no control over gene expression. A large number ofpromoters, recognized by a variety of potential host cells, are wellknown.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Additional promoters which may be of interest include, but are notlimited to: SV40 early promoter (Benoist and Chambon, 1981, Nature290:304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad.U.S.A. 81:659-663); the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797);herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.Sci. U.S.A. 78:1444-1445); promoter and regulatory sequences from themetallothionine gene Prinster et al., 1982, Nature 296:39-42); andprokaryotic promoters such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.75:3727-3731); or the tac promoter (DeBoer et al., 1983, Proc. Natl.Acad. Sci. U.S.A. 80:21-25). Also of interest are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315:115-122); the immunoglobulin gene control region thatis active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658;Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol.Cell. Biol. 7:1436-1444); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-495); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science253:53-58); the alpha 1-antitrypsin gene control region that is activein liver (Kelsey et al., 1987, Genes and Devel. 1:161-171); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315:338-340; Kollias et al., 1986, Cel146:89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712); the myosin light chain-2 gene control region that is activein skeletal muscle (Sani, 1985, Nature 314:283-286); and thegonadotropic releasing hormone gene control region that is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

An enhancer sequence may be inserted into the vector to increasetranscription by higher eukaryotes. Enhancers are cis-acting elements ofDNA, usually about 10-300 bp in length, that act on the promoter toincrease transcription. Enhancers are relatively orientation andposition independent, having been found at positions both 5′ and 3′ tothe transcription unit. Several enhancer sequences available frommammalian genes are known (e.g., globin, elastase, albumin,alpha-feto-protein and insulin). Typically, however, an enhancer from avirus is used. The SV40 enhancer, the cytomegalovirus early promoterenhancer, the polyoma enhancer, and adenovirus enhancers known in theart are exemplary enhancing elements for the activation of eukaryoticpromoters. While an enhancer may be positioned in the vector either 5′or 3′ to a coding sequence, it is typically located at a site 5′ fromthe promoter. A sequence encoding an appropriate native or heterologoussignal sequence (leader sequence or signal peptide) can be incorporatedinto an expression vector, to promote extracellular secretion of theIL-2 mutein or IL-2 mutein Fc-fusion protein. The choice of signalpeptide or leader depends on the type of host cells in which the proteinis to be produced, and a heterologous signal sequence can replace thenative signal sequence. Examples of signal peptides that are functionalin mammalian host cells include the following: the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., 1984,Nature 312:768; the interleukin-4 receptor signal peptide described inEP Patent No. 0367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; the type II interleukin-1 receptorsignal peptide described in EP Patent No. 0 460 846.

The vector may contain one or more elements that facilitate expressionwhen the vector is integrated into the host cell genome. Examplesinclude an EASE element (Aldrich et al. 2003 Biotechnol Prog.19:1433-38) and a matrix attachment region (MAR). MARs mediatestructural organization of the chromatin and may insulate the integratedvector from “position” effect. Thus, MARs are particularly useful whenthe vector is used to create stable transfectants. A number of naturaland synthetic MAR-containing nucleic acids are known in the art, e.g.,U.S. Pat. Nos. 6,239,328; 7,326,567; 6,177,612; 6,388,066; 6,245,974;7,259,010; 6,037,525; 7,422,874; 7,129,062.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe flanking sequences described herein are not already present in thevector, they may be individually obtained and ligated into the vector.Methods used for obtaining each of the flanking sequences are well knownto one skilled in the art.

After the vector has been constructed and a nucleic acid moleculeencoding an IL-2 mutein or IL-2 mutein Fc-fusion protein has beeninserted into the proper site of the vector, the completed vector may beinserted into a suitable host cell for amplification and/or polypeptideexpression. The transformation of an expression vector into a selectedhost cell may be accomplished by well known methods includingtransfection, infection, calcium phosphate co-precipitation,electroporation, microinjection, lipofection, DEAE-dextran mediatedtransfection, or other known techniques. The method selected will inpart be a function of the type of host cell to be used. These methodsand other suitable methods are well known to the skilled artisan, andare set forth, for example, in Sambrook et al., 2001, supra.

A host cell, when cultured under appropriate conditions, synthesizes anIL-2 mutein or IL-2 mutein Fc-fusion protein that can subsequently becollected from the culture medium (if the host cell secretes it into themedium) or directly from the host cell producing it (if it is notsecreted). The selection of an appropriate host cell will depend uponvarious factors, such as desired expression levels, polypeptidemodifications that are desirable or necessary for activity (such asglycosylation or phosphorylation) and ease of folding into abiologically active molecule. A host cell may be eukaryotic orprokaryotic.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, immortalized cell linesavailable from the American Type Culture Collection (ATCC) and any celllines used in an expression system known in the art can be used to makethe recombinant polypeptides of the invention. In general, host cellsare transformed with a recombinant expression vector that comprises DNAencoding a desired IL-2 mutein or IL-2 mutein Fc-fusion. Among the hostcells that may be employed are prokaryotes, yeast or higher eukaryoticcells. Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Higher eukaryotic cells include insect cellsand established cell lines of mammalian origin. Examples of suitablemammalian host cell lines include the COS-7 line of monkey kidney cells(ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells,C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells,or their derivatives such as Veggie CHO and related cell lines whichgrow in serum-free media (Rasmussen et al., 1998, Cytotechnology 28:31), HeLa cells, BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cellline derived from the African green monkey kidney cell line CVI (ATCCCCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821, humanembryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermalA431 cells, human Colo205 cells, other transformed primate cell lines,normal diploid cells, cell strains derived from in vitro culture ofprimary tissue, primary explants, HL-60, U937, HaK or Jurkat cells.Optionally, mammalian cell lines such as HepG2/3B, KB, NIH 3T3 or S49,for example, can be used for expression of the polypeptide when it isdesirable to use the polypeptide in various signal transduction orreporter assays.

Alternatively, it is possible to produce the polypeptide in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Suitableyeasts include Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces strains, Candida, or any yeast strain capable ofexpressing heterologous polypeptides. Suitable bacterial strains includeEscherichia coli, Bacillus subtilis, Salmonella typhimurium, or anybacterial strain capable of expressing heterologous polypeptides. If thepolypeptide is made in yeast or bacteria, it may be desirable to modifythe polypeptide produced therein, for example by phosphorylation orglycosylation of the appropriate sites, in order to obtain thefunctional polypeptide. Such covalent attachments can be accomplishedusing known chemical or enzymatic methods.

The polypeptide can also be produced by operably linking the isolatednucleic acid of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBac® kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), and Luckow and Summers,Bio/Technology 6:47 (1988). Cell-free translation systems could also beemployed to produce polypeptides using RNAs derived from nucleic acidconstructs disclosed herein. Appropriate cloning and expression vectorsfor use with bacterial, fungal, yeast, and mammalian cellular hosts aredescribed by Pouwels et al. (Cloning Vectors: A Laboratory Manual,Elsevier, New York, 1985). A host cell that comprises an isolatednucleic acid of the invention, preferably operably linked to at leastone expression control sequence, is a “recombinant host cell”.

In certain aspects, the invention includes an isolated nucleic acidicacid encoding a human IL-2 mutein that preferentially stimulates Tregulatory cells and comprises a V91K substitution and an amino acidsequence at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to the amino acid sequence set forth in SEQID NO:1. The isolated nucleic acid may encode any of the exemplary IL-2muteins provided herein.

Also included are isolated nucleic acids encoding any of the exemplaryIL-2 mutein Fc-fusion proteins described herein. In preferredembodiments, the Fc portion of an antibody and the human IL-2 mutein areencoded within a single open-reading frame, optionally with a linkerencoded between the Fc region and the IL-2 mutein.

In another aspect, provided herein are expression vectors comprising theabove IL-2 mutein- or IL-2 mutein Fc-fusion protein-encoding nucleicacids operably linked to a promoter.

In another aspect, provided herein are host cells comprising theisolated nucleic acids encoding the above IL-2 muteins or IL-2 muteinFc-fusion proteins. The host cell may be a prokaryotic cell, such as E.coli, or may be a eukaryotic cell, such as a mammalian cell. In certainembodiments, the host cell is a Chinese hamster ovary (CHO) cell line.

In another aspect, provided herein are methods of making a human IL-2mutein. The methods comprising culturing a host cell under conditions inwhich a promoter operably linked to a human IL-2 mutein is expressed.Subsequently, the human IL-2 mutein is harvested from said culture. TheIL-2 mutein may be harvested from the culture media and/or host celllysates.

In another aspect, provided herein are methods of making a human IL-2mutein Fc-fusion protein. The methods comprising culturing a host cellunder conditions in which a promoter operably linked to a human IL-2mutein Fc-fusion protein is expressed. Subsequently, the human IL-2mutein Fc-fusion protein is harvested from said culture. The human IL-2mutein Fc-fusion protein may be harvested from the culture media and/orhost cell lysates.

Pharmaceutical Compositions

In some embodiments, the invention provides a pharmaceutical compositioncomprising a therapeutically effective amount of an IL-2 mutein togetherwith a pharmaceutically effective diluents, carrier, solubilizer,emulsifier, preservative, and/or adjuvant. In certain embodiments, theIL-2 mutein is within the context of an IL-2 mutein Fc-fusion protein.Pharmaceutical compositions of the invention include, but are notlimited to, liquid, frozen, and lyophilized compositions.

Preferably, formulation materials are nontoxic to recipients at thedosages and concentrations employed. In specific embodiments,pharmaceutical compositions comprising a therapeutically effectiveamount of an IL-2 mutein containing therapeutic molecule, e.g, an IL-2mutein Fc-fusion, are provided.

In certain embodiments, the pharmaceutical composition may containformulation materials for modifying, maintaining or preserving, forexample, the pH, osmolarity, viscosity, clarity, color, isotonicity,odor, sterility, stability, rate of dissolution or release, adsorptionor penetration of the composition. In such embodiments, suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine, proline, or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. See,REMINGTON'S PHARMACEUTICAL SCIENCES, 18″ Edition, (A. R. Genrmo, ed.),1990, Mack Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantigen binding proteins of the invention. In certain embodiments, theprimary vehicle or carrier in a pharmaceutical composition may be eitheraqueous or non-aqueous in nature. For example, a suitable vehicle orcarrier may be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryvehicles. In specific embodiments, pharmaceutical compositions compriseTris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5,and may further include sorbitol or a suitable substitute therefor. Incertain embodiments of the invention, II-2 mutein compositions may beprepared for storage by mixing the selected composition having thedesired degree of purity with optional formulation agents (REMINGTON'SPHARMACEUTICAL SCIENCES, supra) in the form of a lyophilized cake or anaqueous solution. Further, in certain embodiments, the IL-2 muteinproduct may be formulated as a lyophilizate using appropriate excipientssuch as sucrose.

The pharmaceutical compositions of the invention can be selected forparenteral delivery. Alternatively, the compositions may be selected forinhalation or for delivery through the digestive tract, such as orally.Preparation of such pharmaceutically acceptable compositions is withinthe skill of the art. The formulation components are present preferablyin concentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

When parenteral administration is contemplated, the therapeuticcompositions for use in this invention may be provided in the form of apyrogen-free, parenterally acceptable aqueous solution comprising thedesired IL-2 mutein composition in a pharmaceutically acceptablevehicle. A particularly suitable vehicle for parenteral injection issterile distilled water in which the IL-2 mutein composition isformulated as a sterile, isotonic solution, properly preserved. Incertain embodiments, the preparation can involve the formulation of thedesired molecule with an agent, such as injectable microspheres,bio-erodible particles, polymeric compounds (such as polylactic acid orpolyglycolic acid), beads or liposomes, that may provide controlled orsustained release of the product which can be delivered via depotinjection. In certain embodiments, hyaluronic acid may also be used,having the effect of promoting sustained duration in the circulation. Incertain embodiments, implantable drug delivery devices may be used tointroduce the IL-2 mutein composition.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving IL-2 mutein compositions insustained- or controlled-delivery formulations. Techniques forformulating a variety of other sustained- or controlled-delivery means,such as liposome carriers, bio-erodible microparticles or porous beadsand depot injections, are also known to those skilled in the art. See,for example, International Patent Application No. PCT/US93/00829, whichis incorporated by reference and describes controlled release of porouspolymeric microparticles for delivery of pharmaceutical compositions.Sustained-release preparations may include semipermeable polymermatrices in the form of shaped articles, e.g., films, or microcapsules.Sustained release matrices may include polyesters, hydrogels,polylactides (as disclosed in U.S. Pat. No. 3,773,919 and EuropeanPatent Application Publication No. EP 058481, each of which isincorporated by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed. Mater.Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinylacetate (Langer et al., 1981, supra) or poly-D(−)-3-hydroxybutyric acid(European Patent Application Publication No. EP 133,988). Sustainedrelease compositions may also include liposomes that can be prepared byany of several methods known in the art. See, e.g., Eppstein et al.,1985, Proc. Natl. Acad. Sci. U.S.A. 82:3688-3692; European PatentApplication Publication Nos. EP 036,676; EP 088,046 and EP 143,949,incorporated by reference.

Pharmaceutical compositions used for in vivo administration aretypically provided as sterile preparations. Sterilization can beaccomplished by filtration through sterile filtration membranes. Whenthe composition is lyophilized, sterilization using this method may beconducted either prior to or following lyophilization andreconstitution. Compositions for parenteral administration can be storedin lyophilized form or in a solution. Parenteral compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

Aspects of the invention includes self-buffering IL-2 muteinformulations, which can be used as pharmaceutical compositions, asdescribed in international patent application WO 06138181A2(PCT/US2006/022599), which is incorporated by reference in its entiretyherein.

As discussed above, certain embodiments provide IL-2 muteincompositions, particularly pharmaceutical II-2 mutein Fc-fusionproteins, that comprise, in addition to the IL-2 mutein composition, oneor more excipients such as those illustratively described in thissection and elsewhere herein. Excipients can be used in the invention inthis regard for a wide variety of purposes, such as adjusting physical,chemical, or biological properties of formulations, such as adjustmentof viscosity, and or processes of the invention to improve effectivenessand or to stabilize such formulations and processes against degradationand spoilage due to, for instance, stresses that occur duringmanufacturing, shipping, storage, pre-use preparation, administration,and thereafter.

A variety of expositions are available on protein stabilization andformulation materials and methods useful in this regard, such as Arakawaet al., “Solvent interactions in pharmaceutical formulations,” PharmRes. 8(3): 285-91 (1991); Kendrick et al., “Physical stabilization ofproteins in aqueous solution,” in: RATIONAL DESIGN OF STABLE PROTEINFORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds.Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al.,“Surfactant-protein interactions,” Pharm Biotechnol. 13: 159-75 (2002),each of which is herein incorporated by reference in its entirety,particularly in parts pertinent to excipients and processes of the samefor self-buffering protein formulations in accordance with the currentinvention, especially as to protein pharmaceutical products andprocesses for veterinary and/or human medical uses.

Salts may be used in accordance with certain embodiments of theinvention to, for example, adjust the ionic strength and/or theisotonicity of a formulation and/or to improve the solubility and/orphysical stability of a protein or other ingredient of a composition inaccordance with the invention.

As is well known, ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface and by shieldingcharged and polar groups in the protein and reducing the strength oftheir electrostatic interactions, attractive, and repulsiveinteractions. Ions also can stabilize the denatured state of a proteinby binding to, in particular, the denatured peptide linkages (—CONH) ofthe protein. Furthermore, ionic interaction with charged and polargroups in a protein also can reduce intermolecular electrostaticinteractions and, thereby, prevent or reduce protein aggregation andinsolubility.

Ionic species differ significantly in their effects on proteins. Anumber of categorical rankings of ions and their effects on proteinshave been developed that can be used in formulating pharmaceuticalcompositions in accordance with the invention. One example is theHofineister series, which ranks ionic and polar non-ionic solutes bytheir effect on the conformational stability of proteins in solution.Stabilizing solutes are referred to as “kosmotropic.” Destabilizingsolutes are referred to as “chaotropic.” Kosmotropes commonly are usedat high concentrations (e.g., >1 molar ammonium sulfate) to precipitateproteins from solution (“salting-out”). Chaotropes commonly are used todenture and/or to solubilize proteins (“salting-in”). The relativeeffectiveness of ions to “salt-in” and “salt-out” defines their positionin the Hofineister series.

Free amino acids can be used in IL-2 mutein formulations in accordancewith various embodiments of the invention as bulking agents,stabilizers, and antioxidants, as well as other standard uses. Lysine,proline, serine, and alanine can be used for stabilizing proteins in aformulation. Glycine is useful in lyophilization to ensure correct cakestructure and properties. Arginine may be useful to inhibit proteinaggregation, in both liquid and lyophilized formulations. Methionine isuseful as an antioxidant.

Polyols include sugars, e.g., mannitol, sucrose, and sorbitol andpolyhydric alcohols such as, for instance, glycerol and propyleneglycol, and, for purposes of discussion herein, polyethylene glycol(PEG) and related substances. Polyols are kosmotropic. They are usefulstabilizing agents in both liquid and lyophilized formulations toprotect proteins from physical and chemical degradation processes.Polyols also are useful for adjusting the tonicity of formulations.

Among polyols useful in select embodiments of the invention is mannitol,commonly used to ensure structural stability of the cake in lyophilizedformulations. It ensures structural stability to the cake. It isgenerally used with a lyoprotectant, e.g., sucrose. Sorbitol and sucroseare among preferred agents for adjusting tonicity and as stabilizers toprotect against freeze-thaw stresses during transport or the preparationof bulks during the manufacturing process. Reducing sugars (whichcontain free aldehyde or ketone groups), such as glucose and lactose,can glycate surface lysine and arginine residues. Therefore, theygenerally are not among preferred polyols for use in accordance with theinvention. In addition, sugars that form such reactive species, such assucrose, which is hydrolyzed to fructose and glucose under acidicconditions, and consequently engenders glycation, also is not amongpreferred polyols of the invention in this regard. PEG is useful tostabilize proteins and as a cryoprotectant and can be used in theinvention in this regard.

Embodiments of IL-2 mutein formulations further comprise surfactants.Protein molecules may be susceptible to adsorption on surfaces and todenaturation and consequent aggregation at air-liquid, solid-liquid, andliquid-liquid interfaces. These effects generally scale inversely withprotein concentration. These deleterious interactions generally scaleinversely with protein concentration and typically are exacerbated byphysical agitation, such as that generated during the shipping andhandling of a product.

Surfactants routinely are used to prevent, minimize, or reduce surfaceadsorption. Useful surfactants in the invention in this regard includepolysorbate 20, polysorbate 80, other fatty acid esters of sorbitanpolyethoxylates, and poloxamer 188.

Surfactants also are commonly used to control protein conformationalstability. The use of surfactants in this regard is protein-specificsince, any given surfactant typically will stabilize some proteins anddestabilize others.

Polysorbates are susceptible to oxidative degradation and often, assupplied, contain sufficient quantities of peroxides to cause oxidationof protein residue side-chains, especially methionine.

Consequently, polysorbates should be used carefully, and when used,should be employed at their lowest effective concentration. In thisregard, polysorbates exemplify the general rule that excipients shouldbe used in their lowest effective concentrations.

Embodiments of IL-2 mutein formulations further comprise one or moreantioxidants. To some extent deleterious oxidation of proteins can beprevented in pharmaceutical formulations by maintaining proper levels ofambient oxygen and temperature and by avoiding exposure to light.Antioxidant excipients can be used as well to prevent oxidativedegradation of proteins. Among useful antioxidants in this regard arereducing agents, oxygen/free-radical scavengers, and chelating agents.Antioxidants for use in therapeutic protein formulations in accordancewith the invention preferably are water-soluble and maintain theiractivity throughout the shelf life of a product. EDTA is a preferredantioxidant in accordance with the invention in this regard.

Antioxidants can damage proteins. For instance, reducing agents, such asglutathione in particular, can disrupt intramolecular disulfidelinkages. Thus, antioxidants for use in the invention are selected to,among other things, eliminate or sufficiently reduce the possibility ofthemselves damaging proteins in the formulation.

Formulations in accordance with the invention may include metal ionsthat are protein co-factors and that are necessary to form proteincoordination complexes, such as zinc necessary to form certain insulinsuspensions. Metal ions also can inhibit some processes that degradeproteins. However, metal ions also catalyze physical and chemicalprocesses that degrade proteins.

Magnesium ions (10-120 mM) can be used to inhibit isomerization ofaspartic acid to isoaspartic acid. Ca⁺² ions (up to 100 mM) can increasethe stability of human deoxyribonuclease. Me, Mn⁺², and Zn⁺², however,can destabilize rhDNase. Similarly, Ca⁺² and Sr⁺² can stabilize FactorVIII, it can be destabilized by Mg⁺², Mn⁺² and Zn⁺², Cu⁺² and Fe⁺², andits aggregation can be increased by Al⁺³ ions.

Embodiments of IL-2 mutein formulations further comprise one or morepreservatives. Preservatives are necessary when developing multi-doseparenteral formulations that involve more than one extraction from thesame container. Their primary function is to inhibit microbial growthand ensure product sterility throughout the shelf-life or term of use ofthe drug product. Commonly used preservatives include benzyl alcohol,phenol and m-cresol. Although preservatives have a long history of usewith small-molecule parenterals, the development of protein formulationsthat includes preservatives can be challenging. Preservatives almostalways have a destabilizing effect (aggregation) on proteins, and thishas become a major factor in limiting their use in multi-dose proteinformulations. To date, most protein drugs have been formulated forsingle-use only. However, when multi-dose formulations are possible,they have the added advantage of enabling patient convenience, andincreased marketability. A good example is that of human growth hormone(hGH) where the development of preserved formulations has led tocommercialization of more convenient, multi-use injection penpresentations. At least four such pen devices containing preservedformulations of hGH are currently available on the market. Norditropin(liquid, Novo Nordisk), Nutropin AQ (liquid, Genentech) & Genotropin(lyophilized—dual chamber cartridge, Pharmacia & Upjohn) contain phenolwhile Somatrope (Eli Lilly) is formulated with m-cresol.

Several aspects need to be considered during the formulation anddevelopment of preserved dosage forms. The effective preservativeconcentration in the drug product must be optimized. This requirestesting a given preservative in the dosage form with concentrationranges that confer anti-microbial effectiveness without compromisingprotein stability.

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time for which a preservative is in contact with theprotein, significantly minimizing the associated stability risks. Withliquid formulations, preservative effectiveness and stability should bemaintained over the entire product shelf-life (about 18 to 24 months).An important point to note is that preservative effectiveness should bedemonstrated in the final formulation containing the active drug and allexcipient components.

IL-2 mutein formulations generally will be designed for specific routesand methods of administration, for specific administration dosages andfrequencies of administration, for specific treatments of specificdiseases, with ranges of bio-availability and persistence, among otherthings. Formulations thus may be designed in accordance with theinvention for delivery by any suitable route, including but not limitedto orally, aurally, opthalmically, rectally, and vaginally, and byparenteral routes, including intravenous and intraarterial injection,intramuscular injection, and subcutaneous injection.

Once the pharmaceutical composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,crystal, or as a dehydrated or lyophilized powder. Such formulations maybe stored either in a ready-to-use form or in a form (e.g., lyophilized)that is reconstituted prior to administration. The invention alsoprovides kits for producing a single-dose administration unit. The kitsof the invention may each contain both a first container having a driedprotein and a second container having an aqueous formulation. In certainembodiments of this invention, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are provided.

The therapeutically effective amount of an IL-2 mutein-containingpharmaceutical composition to be employed will depend, for example, uponthe therapeutic context and objectives. One skilled in the art willappreciate that the appropriate dosage levels for treatment will varydepending, in part, upon the molecule delivered, the indication forwhich the IL-2 mutein is being used, the route of administration, andthe size (body weight, body surface or organ size) and/or condition (theage and general health) of the patient. In certain embodiments, theclinician may titer the dosage and modify the route of administration toobtain the optimal therapeutic effect. A typical dosage may range fromabout 0.1 μg/kg to up to about 1 mg/kg or more, depending on the factorsmentioned above. In specific embodiments, the dosage may range from 0.5μg/kg up to about 100 μg/kg, optionally from 2.5 μg/kg up to about 50μg/kg.

A therapeutic effective amount of an IL-2 mutein preferably results in adecrease in severity of disease symptoms, in an increase in frequency orduration of disease symptom-free periods, or in a prevention ofimpairment or disability due to the disease affliction.

Pharmaceutical compositions may be administered using a medical device.Examples of medical devices for administering pharmaceuticalcompositions are described in U.S. Pat. Nos. 4,475,196; 4,439,196;4,447,224; 4,447, 233; 4,486,194; 4,487,603; 4,596,556; 4,790,824;4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163,all incorporated by reference herein.

Methods of Treating Autoimmune or Inflammatory Disorders

In certain embodiments, an IL-2 mutein of the invention is used to treatan autoimmune or inflammatory disorder. In preferred embodiments, anIL-2 mutein Fc-fusion protein is used.

Disorders that are particularly amenable to treatment with IL-2 muteindisclosed herein include, but are not limited to, inflammation,autoimmune disease, atopic diseases, paraneoplastic autoimmune diseases,cartilage inflammation, arthritis, rheumatoid arthritis, juvenilearthritis, juvenile rheumatoid arthritis, pauciarticular juvenilerheumatoid arthritis, polyarticular juvenile rheumatoid arthritis,systemic onset juvenile rheumatoid arthritis, juvenile ankylosingspondylitis, juvenile enteropathic arthritis, juvenile reactivearthritis, juvenile Reiter's Syndrome, SEA Syndrome (Seronegativity,Enthesopathy, Arthropathy Syndrome), juvenile dermatomyositis, juvenilepsoriatic arthritis, juvenile scleroderma, juvenile systemic lupuserythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis,polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis,ankylosing spondylitis, enteropathic arthritis, reactive arthritis,Reiter's Syndrome, SEA Syndrome (Seronegativity, Enthesopathy,Arthropathy Syndrome), dermatomyositis, psoriatic arthritis,scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis,polyarteritis nodossa, Wegener's granulomatosis, arteritis, ploymyalgiarheumatica, sarcoidosis, sclerosis, primary biliary sclerosis,sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis,guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermicpsoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus,Still's disease, Systemic Lupus Erythematosus (SLE), myasthenia gravis,inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis,celiac disease, multiple sclerosis (MS), asthma, COPD, rhinosinusitis,rhinosinusitis with polyps, eosinophilic esophogitis, eosinophilicbronchitis, Guillain-Barre disease, Type I diabetes mellitus,thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud'sphenomenon, autoimmune hepatitis, GVHD, transplantation rejection,kidney damage, hepatitis C-induced vasculitis, spontaneous loss ofpregnancy, and the like.

In preferred embodiments, the autoimmune or inflammatory disorder islupus, graft-versus-host disease, hepatitis C-induced vasculitis, Type Idiabetes, multiple sclerosis, spontaneous loss of pregnancy, atopicdiseases, and inflammatory bowel diseases.

Methods of Expanding Treg Cells

The IL-2 mutein or IL-2 mutein Fc-fusion proteins may be used to expandTreg cells within a subject or sample. Provided herein are methods ofincreasing the ratio of Tregs to non-regulatory T cells. The methodcomprises contacting a population of T cells with an effective amount ofa human IL-2 mutein or IL-2 mutein Fc-fusion. The ratio may be measuredby determining the ratio of CD3+FOXP3+ cells to CD3+FOXP3− cells withinthe population of T cells. The typical Treg frequency in human blood is5-10% of total CD4+CD3+ T cells, however, in the diseases listed abovethis percentage may be lower or higher. In preferred embodiments, thepercentage of Treg increases at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, at least 200%, at least 300%, at least 400%,at least 500%, at least 600%, at least 700%, at least 800%, at least900%, or at least 1000%. Maximal fold increases in Treg may vary forparticular diseases; however, a maximal Treg frequency that might beobtained through IL-2 mutein treatment is 50% or 60% of total CD4+CD3+ Tcells. In certain embodiments, the IL-2 mutein or IL-2 mutein Fc-fusionprotein is administered to a subject and the ratio of regulatory T cells(Tregs) to non-regulatory T cells within peripheral blood of a subjectincreases.

Because the IL-2 mutein and IL-2 mutein Fc-fusion proteinspreferentially expand Tregs over other cell types, they also are usefulfor increasing the ratio of regulatory T cells (Tregs) to natural killer(NK) cells within the peripheral blood of a subject. The ratio may bemeasured by determining the ratio of CD3+FOXP3+ cells to CD16+ and/orCD56+ lymphocytes that are CD19− and CD3−.

It is contemplated that the IL-2 muteins or IL-2 mutein Fc-fusionproteins may have a therapeutic effect on a disease or disorder within apatient without significantly expanding the ratio of Tregs tonon-regulatory T cells or NK cells within the peripheral blood of thepatient. The therapeutic effect may be due to localized activity of theIL-2 mutein or IL-2 Fc-fusion protein at the site of inflammation orautoimmunity.

EXAMPLES

The following examples, both actual and prophetic, are provided for thepurpose of illustrating specific embodiments or features of the presentinvention and are not intended to limit its scope.

Example 1 Reducing Number of Mutations that Confer High Affinity forCD25

IL-2 muteins with elevated affinity for CD25 and reduced signalingstrength through IL-2Rβγ preferentially promote Treg growth andfunction. To reduce the potential immunogenicity, the minimum number ofmutations required to achieve high affinity for CD25 was sought. Thecrystal structure of IL-2 in complex with its three receptors (PDBcode—2B51) shows V69A and 074P are located in the helical structure thatinteracts with CD25. This may explain why V69A and 074P were frequentlyisolated in two independent IL-2 mutagenesis screens for high CD25binding affinity (Rao et al. 2005; Thanos et al. 2006). This Exampleexplores which of the other mutations in IL-2 mutein “2-4” identified inthe screen of Rao et al. are most important to increase the affinityabove that observed with V69A and 074P alone. The following proteinswere screened by flow cytometry for binding to CD25 on the surface ofactivated T cells. All constructs also included a C-terminal FLAG andpoly-His tag for purification and detection. The specific mutations areprovided in parenthesis.

HaMut1D (V69A, Q74P, N88D, C125A) (SEQ ID NO: 8)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLTHaMut2D(N30S, V69A, Q74P, N88D, C125A) (SEQ ID NO: 9)APTSSSTKKTQLQLEHLLLDLQMILNGINSYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLTHaMut3D(K35R, V69A, Q74P, N88D, C125A) (SEQ ID NO: 10)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPRLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLTHaMut4D(T37A, V69A, Q74P, N88D, C125A) (SEQ ID NO: 11)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLARMLTFKFYMPKKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLTHaMut5D (K48E, V69A, Q74P, N88D, C125A) (SEQ ID NO: 12)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPEKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLTHaMut6D (E68D, V69A, Q74P, N88D, C125A) (SEQ ID NO: 13)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEDALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLTHaMut7D (N71R, V69A, Q74P, N88D, C125A) (SEQ ID NO: 14)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLTHaMut8D(K35R, K48E, E68D, N88D, C125A) (SEQ ID NO: 15)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPRLTRMLTFKFYMPEKATELKHLQCLEEELKPLEDVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQ SIISTLT

HaMut7D bound CD25 with nearly the same affinity as the original isolate“2-4” (^(˜)200 pM), indicating that mutation N71R was capable of greatlyincreasing the affinity above that observed with V69A, Q74P alone(HaMut1D, ^(˜)2 nM). The other constructs possessed affinities similarto or slightly higher than HaMut1D, with the exception of HaMut8D whoseaffinity was only slightly higher than that of WT IL-2.

Example 2 Il-2 Muteins Fused to IgG1-Fc Domains for Improved Half-Life

To reduce the dosing frequency required to achieve Treg enrichment withan IL-2 mutein, we evaluated various fusions between IL-2 and IgG1-Fcdomains. The Fc domains contained point mutations to abolish effectorfunctions mediated by IgG1, such as target cell lysis. The Fc effectorfunction mutations utilized in our studies were either A327Q, Ala Ala(L234A+L235A) or N297G. Because the Treg-selective IL-2 muteins havepartial reduction in IL-2 potency, it was important to fuse IL-2 to Fcin such a way that did not significantly impact IL-2R signaling. Thus,IL-2 muteins were tested for IL-2R activation with and without Fcfusion.

To determine if IL-2 dimerization by Fc fusion would increase IL-2Rsignaling strength due to increased avidity for IL-2R, a weaker IL-2mutein (haD5) (US20110274650) was fused to the amino terminus of Fc,separated by a GGGGS (SEQ ID NO: 5) linker sequence. This muteinpossessed 3 mutations impacting IL-2R signaling (E15Q, H₁₆N, N88D), 8mutations to confer high affinity for CD25 (N29S, Y31H, K35R, T37A,K48E, V69A, N71R, Q74P) (Rao et al. 2005), and C125S to prevent cysteinemispairing and aggregation. Fusion to Fc in this manner completelyabrogated the biological activity of haD5, while its high-affinitybinding to cell surface CD25 was enhanced, likely due to increasedavidity from dimerization.

IL-2 muteins were also fused to either the N- or C-terminus of an Fcheterodimer, such that only one chain of the Fc dimer bore the IL-2domain. Heterodimeric pairing between two asymmetric Fc chains waspromoted by electrostatic interactions between introduced lysines on oneFc chain and introduced aspartic acids on the other Fc chain. IL-2mutein haD6 was fused to the N-terminus of one Fc chain or the other, inthe event that one configuration was preferred, resulting in two proteinconstructs termed haD6.FcDD and haD6.FcKK. We also fused mutein haMut7Dto the C-terminus of the Fc heterodimer with one or two GGGGS (SEQ IDNO: 5) linkers (FcKK(G4S)haMut7D, FcKK(G4S)2haMut7D). Fusion of the IL-2mutein haD6 to the N-terminus of the Fc heterodimer resulted in apartial loss of activity relative to free haD6 in both pSTAT5 and T cellproliferation experiments. In contrast, fusion of haMut7D to theC-terminus of the Fc heterodimer with either one or two GGGGS (SEQ IDNO: 5) linkers did not alter the potency of haMut7D.

Fusion of an IL-2 mutein to the C-terminus of an Fc homodimer was alsoinvestigated. Total PBMC were activated in T75 tissue culture flasks at300 million cells per 100 ml with 100 ng/ml anti-CD3 (OKT3). On day 3 ofculture, cells were washed 3 times and rested in fresh media for 3 days.Cells were then stimulated with IL-2 variants at 10× dose titrationranging from 1 μM to 10 nM at a final volume of 50 μl. The level ofSTAT5 phosphorylation was measured using BD phosflow buffer kit.Briefly, 1 ml of BD lyse/fix phosflow buffer was added to stopstimulation. Cells were fixed for 20 min at 37° C. and permeabilizedwith 1×BD phosflow perm buffer on ice before stained for CD4, CD25,FOXP3 and pSTAT5.

As can be seen in FIG. 1, the bioactivity of muteins haMut1D and haMut7Dwas not altered by fusion to the C-terminus of an Fc homodimer. Thus,fusion between the N-terminus of IL-2 and C-terminus of Fc did notcompromise the agonist activity of the IL-2 muteins, even in the contextof an Fc.IL-2 homodimer. In these constructs, the C125A mutation wasused in place of C1255 for improved manufacturing.

Example 3 Tuning IL-2 Mutein Potency to Achieve Preferential Treg Growth

The initial panel of IL-2 muteins contained N88D alone or with 1 or 2additional mutations impacting IL-2R signaling. A second panel ofmuteins was designed, all with single point mutations, with the goal ofidentifying muteins with either similar or slightly more potent agonismthan those of the N88D series. A panel of 24 signaling mutations wasidentified based on predicted IL-2Rβ-interacting amino acids (crystalstructure, PDB code—2B51). Particular substitutions were selected basedon predicted decrease in the binding free energy between the mutein andIL-2Rβ. The binding free energy was calculated using EGAD computationalalgorithm (Handel's Laboratory, University of California at San Diego,USA). The binding free energy of a mutant is defined asΔΔG_(mut)=μ(ΔG_(mut)−ΔG_(wt)). Where, μ(=0.1, in general) is the scalingfactor used to normalize the predicted changes in binding affinity tohave a slope of 1 when comparing with the experimental energies (Pokalaand Handel 2005). The free energy of dissociation (ΔG) was defined asthe energy difference between the complex (ΔG_(bound)) and free states(ΔG_(free)). The dissociation energy ΔGmut was calculated for eachsubstitution.

A panel of IL-2 muteins with the following substitutions (H16E, H16Q,L19K, D₂₀R, D20K, D20H, D20Y, M23H, D84K, D84H, 587Y, N88D, N88K, N881,N88H, N88Y, V91N, V91K, V91H, V91R, I92H, E95K, E95R, or E951) wasexpressed as C-terminal fusions to the Fc heterodimer. These constructsalso contained the haMut7 mutations for high CD25 binding affinity(V69A, N71R, Q74P) and C125A for efficient folding.

The panel was screened for potency in the T cell STAT5 phosphorylationassay of Example 2, and H16E, D84K, V91N, V91K, and V91R were found topossess activity less than wild type IL-2 and more than N88D (FIG. 2).

H16E, D84K, V91N, V91K, and V91R possessed activity less than wild typeIL-2 and more than N88D.

Selected muteins were also tested in T cell and NK growth assays.

For the T-cell assay, total PBMCs were activated at 3 million/ml with100 ng OKT3. On day 2, cells were washed 3 times and rested in freshmedia for 5 days. Cells were then labeled with CFSE and further culturedin a 24 well plate at 0.5 million/well in IL-2 containing media for 7days before FACS analysis. The proliferation of T cell subsets ispresented in FIG. 3 as CFSE dilution (median CFSE fluorescence).

For the NK-cell assay, MACS sorted CD16+ NK cells were cultured in IL-2containing media for 3 days at 0.1 million/well in 96 well plates. 0.5μCi³H-thymidine was added to each well during the final 18 hours ofincubation. The results are shown in FIG. 4.

Mutants H16E, D84K, V91N, V91K, and V91R mutants were capable ofstimulating Treg growth similar to WT IL-2 but were approximately 10×less potent on other T cells (FIG. 3), and approximately 100× lesspotent on NK cells (FIG. 4).

A separate panel of Fc.IL-2 fusion proteins was designed in which thedistance between the Fc heterodimer and the mutein haMut7 (V69A, N71R,Q74P, C125A) was reduced by a series of individual amino acidtruncations.

(SEQ ID NO: 22)

(SEQ ID NO: 23)    

(SEQ ID NO: 24)    

(SEQ ID NO: 25)    

(SEQ ID NO: 26)    

(SEQ ID NO: 27)    

(SEQ ID NO: 28)    

(SEQ ID NO: 29)    

(SEQ ID NO: 30)    

Trunc1-Trunc4 possessed potency equal to the full length parentconstruct Fc.haMut7 as measured by STAT5 phosphorylation and by T celland NK cell proliferation as described for FIGS. 2, 3, and 4. Trunc5 andTrunc6 stimulated weaker responses yet stronger than those stimulated bythe N88D mutation (haD and haMut7D) and very similar to those stimulatedby V91K. Trunc7 was weaker than N88D muteins, and Trunc8 had very littleactivity. When tested on NK cells, however, Trunc5 and Trunc6 werestronger agonists than V91K, indicating that Treg selectivity was morereadily achieved with signaling mutations rather than steric hindranceby a proximal Fc domain.

Example 4 High CD25 Affinity Mutations in the Context of an Fc Homodimer

The mutations that conferred high CD25 binding affinity were consideredadvantageous because they increased tropism for CD25-high T cells, andbecause they promoted long term CD25::IL-2mutein association andprolonged signaling. However, reducing mutation number may reduceimmunogenicity potential. The N88D or the V91K muteins, with and withoutthe haMut1 high affinity mutations V69A and 074P, were expressed asfusions to the C-terminus of an Fc homodimer and compared forbioactivty. In pSTAT5 stimulation assays, the homodimerization had noeffect on signal strength relative to monomeric mutein. The reversion ofthe high affinity mutations V69A and 074P also did not affect pSTAT5signaling. In T cell growth assays, the high affinity mutations reducedactivity on conventional CD4 T cells and CD8 T cells but not onregulatory T cells (FIG. 5). The high affinity mutations also did notalter proliferative responses in NK cells (FIG. 6).

To determine if the high affinity mutations impacted T cell responses invivo, we dosed humanized mice (NOD.SCID.II2rg-null mice reconstitutedwith human CD34+ hematopoietic stem cells) with the Fc.IL-2 muteinfusion proteins and monitored Treg expansion. Seven week oldNOD.SCID.II2rg-null (NSG) mice (Jackson Labs, Bar Harbor, Me.) wereirradiated (180 rad) and reconstituted with 94,000 human fetal liverCD34⁺ hematopoietic stem cells. At 21 weeks, mice were distributed into6 groups based on equal distribution of percent chimerism (determined byflow cytometry of PBL) and were given 1 μg sub-cutaneous injections ofthe indicated Fc.mutein fusion proteins or PBS on day 0 and day 7. Onday 11, T cell subset frequencies in blood were determined by flowcytometry. At the low dose of 1 μg per animal, the high affinitymutations did not improve Treg expansion beyond that observed with theN88D or V91K mutations alone (FIG. 7).

Treg expansion was selective in that FOXP3⁻CD4⁺ T cells did not increasein abundance relative to total peripheral blood leukocytes (PBL) whichincludes a mixture of human B and T cells, and mouse myeloid cells.Furthermore, at higher doses, the high affinity mutations promoted anincrease in CD25⁺FOXP3⁻ T cells, thus reducing Treg selectivity. Thus,in the context of the Fc homodimer, the high affinity mutations were notconsidered necessary for promoting preferential Treg growth.

Fc.WT IgG1Fc(N297G_delK)::G4S::hulL-2(C125A) (SEQ ID NO: 16)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG GGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT Fc.haMut1V91KIgG1Fc(N297G_delK)::G4S::hulL-2 (V69A, Q74P, V91K, C125A)(SEQ ID NO: 17) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG GGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT Fc.V91KIgG1Fc(N297G_delK)::G4S::hulL-2(V91K, C125A) (SEQ ID NO: 18)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG GGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINKIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT Fc.haMut1N88DIgG1Fc(N297G_delK)::G4S::hulL-2 (V69A, Q74P, N88D, C125A)(SEQ ID NO: 19) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG GGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT Fc.N88DIgGlFc(N297G_delK)::G4S::hulL-2(N88D, C125A) (SEQ ID NO: 20)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG GGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

Example 5 Prolonged Cell Surface CD25 Association of Fc.IL-2 Muteins

An unexpected result from the humanized mouse studies was that, despitetheir reduced signaling capacity, the muteins induced more robust Tregenrichment relative to Fc.WT IL-2. Greater Treg enrichment and FOXP3upregulation relative to that seen with Fc.WT was observed at a dose of1 μg/mouse (FIG. 7) and at a lower dose of 0.5 μg/mouse (FIG. 8). Thisincreased potency in vivo may have resulted from reduced consumption byT cells, making more Fc.IL-2 mutein available for prolonged signaling.

In vitro and in vivo PK studies failed, however, to demonstratesignificantly increased persistence of Fc.V91K or Fc.N88D relative toFc.WT in supernatants from activated T cell cultures or serum from dosedmice. Because the Fc fusions bore two IL-2 mutein domains, we reasonedthat increased endosomal recycling may result in prolonged cell surfaceassociation due to increased avidity for CD25. Indeed, we found thatFc.V91K and Fc.N88D persisted more efficiently than Fc.WT on the surfaceof previously activated T cells following a brief exposure the fusionproteins (FIG. 9).

Primary PBMCs were prestimulated for 2 days with 100 ng/ml OKT3. Cellswere harvested, washed 4 times and rested for overnight in media. Cellswere then pulsed with 400 μM Fc.IL-2 for 30 min at 37° C. After pulse,cells were either harvested for T0 after one wash, or washed anadditional 3 times in 12 ml of warm media and cultured for 4 hours. Todetect cell-associated Fc.IL-2, cells were stained with anti-humanIgG-FITC (Jackson Immunoresearch) and anti-CD25-APC.

Example 6 Fusion Sequence Optimization

In preclinical studies in mice, our Fc.IL-2 muteins showed differentialexposure when serum concentrations of the intact molecule were comparedthat of the human Fc portion only, indicative of circulating human Fccatabolite. To optimize the in vivo stability and pharmacokinetics ofour Fc.IL-2 muteins, we characterized fusion sequence modifications fortheir impact on protoeolytic degradation of Fc.IL-2 muteins in systemiccirculation and during recycling through the reticuloendothelial system.

The following constructs were evaluated for proteolytic degradation invitro and in viva

(SEQ ID NO: 31) (Ala_Ala)_G4S

(SEQ ID NO: 32) (N297G_delK)_G4S

(SEQ ID NO: 33) (N297G_KtoA)_AAPT

(SEQ ID NO: 34) (N297G_KtoA)_AAPA

Stability was measured by quantitative immunoassays comparingconcentrations over time of total human Fc to that of intact Fc.IL-2mutein. Proteolysis of Fc.IL-2 muteins was verified by western blotanalysis utilizing anti-IL-2 and anti-human Fc antibodies, followed byimmunocapture of catabolites and characterization by mass spectrometry.Characterization by mass spectrometry of catabolites of (Ala_Ala)_G4Sfrom in vitro and in vivo samples identified the C-terminal Lys of theFc domain as a proteolytic cleavage site. Deletion or mutation of theC-terminal lysine of the Fc domain ((N297GdelK)_G4S and(N297G_KtoA)_AAPT) resulted in prolonged in vitro stability in mouseserum at 37° C. compared to Fc constructs with the C-terminal lysine((Ala_Ala)_G4S). This prolonged in vitro serum stability translated togreater exposure in mice as measured by the area under the Fc.IL-2mutein serum concentration versus time curve (AUC). This prolongedstability of Fc.IL-2 muteins lacking the C-terminal Fc lysine was alsoobserved in vitro in serum from cynomolgus monkeys and humans. Mutationof Thr-3 of IL-2 to Ala ((N297G_KtoA)_AAPA) resulted in decreased invitro stability at 37° C. (compared to (N297G_KtoA)_AAPT) in mouse serumand in separate incubations with recombinant human cathepsin D and L.This decreased in vitro serum stability translated to lower exposure(AUC) in mice in vivo for (N297G_KtoA)_AAPA compared to(N297G_KtoA)_AAPT. Characterization of catabolites of (N297G_KtoA)_AAPAfrom in vitro and in vivo samples by mass spectrometry identified Lys 8and Lys 9 of the IL-2 mutein domain as residues susceptible toproteolysis which was not observed for equivalent samples of(N297G_KtoA)_AAPT. Decreased stability at 37° C. of (N297G_KtoA)_AAPA tothat of (N297G_KtoA)_AAPT was also observed in vitro in serum fromcynomolgus monkeys and humans.

Because of the importance of glycosylation in this region, and topotentially improve upon the manufacturability of the fusion protein,the fusion sequences were altered to promote N-linked rather thanO-linked glycosylation, as follows.

Original IgG1Fc(N297G_delK)::G4S::hulL-2(V91K,C125A) (SEQ ID NO: 32)

Altered IgG1Fc(N297G_delK)::G4S::hulL-2(T3N,V91K,C125A) (SEQ ID NO: 35)

IgG1Fc(N297G_delK)::G4S::hulL-2(T3N,S5T,V91K,C125A) (SEQ ID NO: 36)

IgG1Fc(N297G_delK)::GGNGT::hulL-2(T3A,V91K,C125A) (SEQ ID NO: 37)

IgG1Fc(N297G_delK)::YGNGT::hulL-2(T3A,V91K,C125A)TC (SEQ ID NO: 38)

Example 7 Cynomolgus Monkey PK/PD Determination

Standard IL-2 immune stimulating therapies require drug free holidays(no exposure) between dosing cycles to avoid undesirable side effects.In contrast, Treg expansion or stimulation therapies may requireprolonged exposure with sustained trough drug levels (serum C_(min))sufficient for Treg stimulation but with maximal exposures (serumC_(max)) below drug levels that lead to immune activation. This exampledemonstrates dosing strategies of half-life extended muteins incynomolgus monkeys for extended target coverage (serum C_(min)) whilemaintaining maximal exposures (serum C_(max)) below drug levelscontemplated to be necessary for proinflammatory immune activation.

Cynomolgus monkeys are dosed with Fc.V91K(IgG1cFc(N297G_delK)::G4S::hulL-2(V91K, C125A) in four groups (A-D),with three groups (A-C) dosed subcutaneously and one group (D) dosedintravenously. For each group, four biologically naïve male cynomolgusmonkeys are dosed per the dosing strategy outlined below. Subcutaneousdosing of half-life extended muteins may allow for greater lymphaticabsorption resulting in lower maximal exposure (serum C_(max)) and/or amore robust pharmacological response (Treg expansion). Dosing strategyfor group A consists of three consecutive 10 microgram per kilogramdoses on Day 0, 2, and 4 for cycle 1 and 10 microgram per kilogram onDay 14, allowing prolonged target coverage similar to a higher initialdose of 50 microgram per kilogram while maintaining a lower maximalexposure (C_(max)). The dosing strategy for group B is 50 microgram perkilogram dosed on Day 0 and 14 for comparison to Group A. The dosingstrategy for group C is 50 microgram per kilogram dosed on Day 0 and 28.Allowing the determination of whether trough coverage is required forsustaining Treg enrichment or whether a drug free holiday is beneficialbetween dosing cycles. The dosing strategy for the intravenous dosingarm group D is 50 microgram per kilogram dosed on Day 0, allowing acomparison of maximal exposures (C_(max)) and Treg enrichmentdifferences to that of subcutaneous dosing.

Pharmacokinetics (quantitative immunoassay for intact molecule and totalhuman Fc), anti-drug antibodies, shed soluble CD25, and serum cytokines(IL-1β, TNF-α, IFN-γ, IL-10, IL-5, IL-4, and IL-13) are measured at thefollowing time points for each dose group specified:

-   Group A: pre-dose (first cycle; dose 1), 48 (pre-dose first cycle;    dose 2), 96 (pre-dose first cycle; dose 3), 100, 104, 120, 168, 216,    264, 336 (pre-dose second cycle), 340, 344, 360, 408, 456, 504, 576,    672, 744, 840, and 1008 hours.-   Group B: pre-dose (first cycle), 4, 8, 24, 72, 120, 168, 240, 336    (pre-dose second cycle), 340, 344, 360, 408, 456, 504, 576, 672,    744, 840, and 1008 hours.-   Group C: pre-dose (first cycle), 4, 8, 24, 72, 120, 168, 240, 336,    408, 504, 672 (pre-dose second cycle), 676, 680, 696, 744, 792, 840,    912, 1008, 1080, and 1176 hours.-   Group D: pre-dose (first cycle), 0.25, 1, 4, 8, 24, 72, 120, 168,    240, 336, 408, 504, and 672 hours.

Pharmacodynamics (immunopheotyping and enumeration of peripheral bloodTregs, non-regulatory CD4 and CD8 T cells, and NK cells) is measured atthe following time points for each dose group specified:

-   Group A: pre-dose (first cycle; dose 1), 96 (pre-dose first cycle;    dose 3), 168, 336 (pre-dose second cycle), 456, and 576 hours.-   Group B: pre-dose (first cycle), 120, 240, 336 (pre-dose second    cycle), 456, and 576 hours.-   Group C: pre-dose (first cycle), 120, 240, 672 (pre-dose second    cycle), 792, and 912 hours.-   Group D: pre-dose (first cycle), 120 and 240 hours.

Hematology and clinical chemistry are assessed for all animals and dosegroups pre-dose and at 24 hours post initial dose per dose group. Thefollowing parameters are evaluated.

Hematology:

-   -   leukocyte count (total and absolute differential)    -   erythrocyte count    -   hemoglobin    -   hematocrit    -   mean corpuscular hemoglobin, mean corpuscular volume, mean        corpuscular hemoglobin concentration (calculated)    -   absolute reticulocytes    -   platelet count    -   blood cell morphology    -   red cell distribution width    -   mean platelet volume        Clinical Chemistry:    -   alkaline phosphatase    -   total bilirubin (with direct biliru bin if total bilirubin        exceeds 1 mg/dL)    -   aspartate aminotransferase    -   alanine aminotransferase    -   gamma glutamyl transferase    -   urea nitrogen    -   creatinine    -   total protein    -   albumin    -   globulin and A/G (albumin/globulin) ratio (calculated)    -   glucose    -   total cholesterol    -   triglycerides    -   electrolytes (sodium, potassium, chloride)    -   calcium    -   phosphorus

Example 8 Aglycosylated IgG1 Fc

Naturally occurring IgG antibodies posses a glycosylation site in theconstant domain 2 of the heavy chain (CH2). For example, human IgG1antibodies have a glycosylation site located at the position Asn297 (EUnumbering). To date, the strategies for making aglycosylated antibodiesinvolve replacing the Asn residue with an amino acid that resembles Asnin terms of physico-chemical properties (e.g., Gln) or with Ala residuewhich mimics the Asn side chain without the polar groups. This Exampledemonstrates the benefits of replacing Asn with Glycine (N297G). N297GFc are aglcosylated molecules with better biophysical properties andmanufacturability attributes (e.g., recovery during purification).

Examination of multiple known crystal structures of Fc fragments and IgGantibodies revealed considerable conformational flexibility around theglycosylated loop segment, particularly at the position Asn297 that isglycosylated. In many of the known crystal structures, Asn297 adaptedpositive backbone dihedral angles. Gly has high propensity to adaptpositive backbone dihedral angle due to the lack of side chain atoms.Therefore, based on this conformation and structure reason, Gly may be abetter replacement for Asn than N297Q or N297A.

Mutating Asn297 with Gly leads to aglcosylated molecules with muchimproved recovery (or efficiency) in the purification process andbiophysical properties. For example, the percentage of recovery (finalyield) from the protein A pool was 82.6% for the N297G mutation,compared to 45.6% for N297Q and 39.6% for N297A. SPHP column analysisrevealed the lower percentage of recovery for the N297Q and N297Amutants was due to a tailing peak, which indicates high molecular weightaggregation and/or misfolded species. This result was re-confirmed at alarger, 2 L scale run.

In the biopharmaceutical industry, molecules with potential need forlarge-scale production, e.g, potential to be sold as a drug, areassessed for a number of attributes to mitigate the risk that themolecule is not amenable to large-scale production and purification. Inthe manufacturability assessments, N297G revealed robustness to pHchanges. N297G had no aggregation issue; whereas N297Q and N297A had 20%and 10% increase in aggregation, respectively. Although N297G had bettermanufacturability attributes, it was similar to N297Q and N297A in allthe functional assays in which it was tested. For example, in ADCCassays, N297G lacked cytotoxicity similarly to N297Q and N297A.

Example 9 Stabilized aglyosylated IgG1 Fc

This Example describes a method of improving stability of IgG antibodyscaffolds by introducing engineered disulfide bond(s). Naturallyoccurring IgG antibodies are stable molecules. However, for sometherapeutic applications, it may be necessary to make mutations orcreate aglycosylated molecules. For example, aglycosylated IgG moleculesmay be used in therapeutic indications where there is a need to avoidADCC and binding to Fcgamma receptors. However, the aglycosylated IgG1has much lower melting temperature (CH2 domain melting temperaturedecreases by about 10° C.; 70° C. to 60° C.) than the glycosylated IgG1.The observed lower melting temperature negatively impacts variousbiophysical properties of the aglycosylated IgG1. For example,aglycosylated IgG1 has increased level of aggregation at low pH comparedto glycosylated IgG1.

In order to engineer disulfide bonds, a structure based method involvingdistance calculation between the C-alpha atoms was initially used toidentify 54 residue pairs in the Fc region for mutation to Cys. These 54sites were further narrowed down to 4 residue pairs (V259C-L306C,R292C-V302C, A287C-L306C, and V323C-1332C). The criteria used included(i) positions within the CH2 domain, (ii) away from loops, turns andcarbohydrates, (iii) away from Fcgamma receptor and FcRn interactionsites, (iv) solvent accessibility (preferred buried positions), etc.

The paired cysteine substitutions were created in the context of theaglycosylated N297G Fc. Non-reduced peptide mapping analysis revealedthat 3 of the 4 engineered sites formed disulfide bond as expected anddesigned in that context. The V259C-L306C mutation did not formdisulfide bond correctly and led to mis-pairing with the nativedisulfide already present in the CH2 domain. The other three designsR292C-V302C, A287C-L306C, and V323C-1332C formed disulfide bondcorrectly as predicted and designed. Adding the disulfide bond to theN297G mutation led to about 15C improvement in thermal stability overthe N297G mutation alone. Of the R292C-V302C, A287C-L306C, andV323C-1332C disulfide variants, R292C-V302C and A287C-L306C had goodpharmacokinetics when administered to rats (t_(1/2) of 11 days and 9days, respectively). This is in contrast to the pharmacokinetics profilewe observed in rats for the previously published CH2 domain disulfidebond (Gong et al., J. Biol. Chem. 2009 284: 14203-14210), which had at_(1/2) of 5 days.

Engineering a disulfide bond in the CH2 domain improves the stability ofthe aglycosylated molecule on par with glycosylated IgG1 molecules (10to 15° C. improvement in the melting temperature as determined byDifferential Scanning calorimetry). The engineered sites describedherein do not lead to disulfide scrambling and the disulfides are formedas predicted in approximately 100% of the population. More importantly,unlike the published disulfide bond site in the CH2 domain, thedisulfide bonds described herein do not impact the rat PK.

What is claimed is:
 1. A polypeptide comprising an Fc region of a humanIgGl antibody wherein said Fc region comprises a N297G mutation, usingEU numbering scheme, one or more substitutions of the amino acidsequence set forth in SEQ ID NO: 3 at position V259, A287, R292, V302,L306, V323, or I332, using EU numbering scheme, with a cysteine aminoacid residue, and said Fc region of a human IgGl comprises at least 95%identity to the amino acid sequence set forth in SEQ ID NO:3.
 2. Thepolypeptide of claim 1, wherein said Fc region comprises a A287C andL306C substitution, using EU numbering scheme, within the amino acidsequence set forth in SEQ ID NO:3.
 3. The polypeptide of claim 1,wherein said Fc region comprises a V259C and L306C substitution, usingEU numbering scheme, within the amino acid sequence set forth in SEQ IDNO:3.
 4. The polypeptide of claim 1, wherein said Fc region comprises aR292C and V302C substitution, using EU numbering scheme, within theamino acid sequence set forth in SEQ ID NO:3.
 5. The polypeptide ofclaim 1, wherein said Fc region comprises a V323C and I332Csubstitution, using EU numbering scheme, within the amino acid sequenceset forth in SEQ ID NO:3.